Abstract

Background

Niemann-Pick type C1 disease (NPC1) is a rare progressive neurodegenerative disorder
caused by mutations in the NPC1 gene. In this lysosomal storage disorder the intracellular
transport and sequestration of several lipids like cholesterol is severely impaired,
resulting in an accumulation of lipids in late endosomes and lysosomes. The neurological
manifestation of the disease is caused by dysfunction and cell death in the central
nervous system. Several animal models were used to analyze the impaired pathways.
However, the underlying pathogenic mechanisms are still not completely understood
and the genetic variability in humans cannot be reflected in these models. Therefore,
a human model using patient-specific induced pluripotent stem cells provides a promising
approach.

Methods

We reprogrammed human fibroblasts from a NPC1 patient and a healthy control by retroviral
transduction with Oct4, Klf4, Sox2 and c-Myc. The obtained human induced pluripotent
stem cells (hiPSCs) were characterized by immunocytochemical analyses. Neural progenitor
cells were generated and patch clamp recordings were performed for a functional analysis
of derived neuronal cells. Filipin stainings and the Amplex Red assay were used to
demonstrate and quantify cholesterol accumulation.

Results

The hiPSCs expressed different stem cell markers, e.g. Nanog, Tra-1-81 and SSEA4.
Using the embryoid body assay, the cells were differentiated in cells of all three
germ layers and induced teratoma in immunodeficient mice, demonstrating their pluripotency.
In addition, neural progenitor cells were derived and differentiated into functional
neuronal cells. Patch clamp recordings revealed voltage dependent channels, spontaneous
action potentials and postsynaptic currents. The accumulation of cholesterol in different
tissues is the main hallmark of NPC1. In this study we found an accumulation of cholesterol
in fibroblasts of a NPC1 patient, derived hiPSCs, and neural progenitor cells, but
not in cells derived from fibroblasts of a healthy individual. These findings were
quantified by the Amplex Red assay, demonstrating a significantly elevated cholesterol
level in cells derived from fibroblasts of a NPC1 patient.

Conclusions

We generated a neuronal model based on induced pluripotent stem cells derived from
patient fibroblasts, providing a human in vitro model to study the pathogenic mechanisms of NPC1 disease.